The aim of this proposal is to identify the immunological mechanisms that provide effective protection against divergent strains of influenza A viruses. Influenza A viruses pose a serious threat to public health and the emergence of H5N1 and H7N9 avian subtypes presents a considerable risk for a deadly pandemic. Vaccination is the most effective strategy for controlling influenza infections, however current vaccines only protect against a small subset of strains. A universal vaccine that induces immunity to proteins conserved across all influenza A viruses would protect against newly emerging and pandemic strains. However, candidate universal vaccines do not induce effective immune responses to conserved influenza A proteins. Thus, to design a successful universal vaccine, we must first understand how to efficiently elicit the components of the immune response that provide immunity to influenza viruses of distinct subtypes. We recently identified an innovative means to enhance immunity against multiple strains of influenza by treating mice with the immunosuppressant, rapamycin, during a primary H3N2 infection. Rapamycin treatment protected mice from subsequent lethal infections with H5N1, H7N9, and H1N1 strains. We will use this system as a tool to identify the components of the immune response that contribute to this remarkable protection against distinct strains of influenza. We hypothesize that the optimal immune response will consist of augmented cross-reactive antibodies in conjunction with an optimized CD8+ T cell response. To test this hypothesis, we propose the following specific aims: 1. Investigate the mechanisms of cross-reactive antibody-mediated protection. Rapamycin enhances generation of cross-reactive antibodies, however the mechanisms of antibody protection and the identity of the viral epitopes they bind are not known. Thus, we will examine how these cross-reactive antibodies protect and identify which antibody specificities correlate with improved protection. 2. Examine molecular and cellular mechanisms of germinal center formation that favor cross-reactive antibodies. While cross-reactive antibodies are present in humans following influenza vaccination or infection, these antibodies are not dominant. Determining how rapamycin alters germinal center formation to increase cross-reactive antibodies will lend insight into methods to enhance the generation of cross-reactive antibodies in humans. 3. Determine the contributions of CD8+ T cells and cross-reactive antibodies in a model of intranasal vaccination. Our preliminary data describe a novel model in which the enhanced immunity to distinct influenza strains is dependent on CD8+ T cells. We will use this model to investigate if cross-reactive antibodies and CD8+ T cells act synergistically to promote optimal heterosubtypic immunity. These experiments will lend valuable insight into the components of the immune response that are critical for protection from divergent influenza infections, which will be essential for creating universal vaccines, and save millions of lives in a deadly pandemic.